This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Characterization of protein folding dynamics from many different perspectives is important in elucidating the kinetic folding mechanisms and the interactions that underlie them. The recent advent of a continuous-flow small-angle X-ray scattering (SAXS) technique allows direct examination of the early compaction events in protein folding. Escherichia coli dihydrofolate reductase (DHFR), a two-subdomain alpha/beta-type protein, as a folding intermediate composed of complex beta-sheet topology, but its overall size and shape have not been characterized. Moreover, observation of the formation process of the DHFR intermediate in the submillisecond time regions has been a challenge in protein folding studies. Here, we performed the continuous-flow SAXS experiment to observe the submillisecond folding dynamics of DHFR in terms of overall size and globularity. The results show that within 300 micro-s of the refolding DHFR forms a compact intermediate with an Rg of 24 A, which is ~1.5-times larger than the Rg of the native state. The Rg of DHFR does not change between 300 micro-s and 10 ms of the refolding reaction, suggesting that the structures and interactions found in the folding intermediate that was previously characterized by stopped-flow experiments are already formed within 300 micro-s. The results may suggest that the beta-sheet contents in native proteins determine the characters of early folding intermediates.
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